Lab 3: Big picture

Where we are in HO-1 purification:

Plasmid Purification and Verification
• Miniprep
• PCR
• Restriction Digest
• DNA gel electrophoresis
Expression
• Transformation MSDS info needed:
• Plating - SOC media
• Expression Culture - Kanamycin
• Cell Harvest - Chloramphenicol
Purification - Tris
• Cell Lysis (and protease inhibition) - Rosetta2 cell line
• Nucleic Acid Precipitation
• Ammonium Sulfate Saturation
• Dialysis/Buffer Exchange
• Ni immobilized metal affinity chromatography
• Anion Exchange
• Verification:
o SDS-Page
o Western Blot
o Bradford microplate

Lab 3 for weeks 5 Transformation and Plating

NOTE: Always Remember to use aseptic technique and wear gloves when handling E. Coli. Anything that touches the
E. Coli should be placed in the Red Biohazard trash bin. Make sure to keep all enzymes and reactions on ice while
prepping. Keep cells on ice until heat shock. Use sterile tubes.

1. Watch the video before coming to the lab.

i. https://www.addgene.org/protocols/bacterial-transformation/
2. Place four LB-Chl/Kan plates into a 37C incubator to warm up.
i. Plating on cold plates could damage cells.
3. Using both a sterile tip and 1.5 mL tube, collect 50 μL of Rosetta II cells.
i. Keep on ice
4. Add 2.5 μL of 10 ng/μL verified plasmid to the cells.
i. Use another group’s if DNA gel was negative.
ii. Cap and flick to mix, do not pipette up and down.
5. Incubate on ice for 15-30 min.
i. This allows time for the plasmid to move into position around the cells.
6. Heat shock in a 42C water bath for 45-60 seconds.
7. Immediately after, place on ice for 2 min.
8. Add 600 μL of SOC broth, cap, and add to flask.
9. Put flask in 37C shaker for 1 hr.
i. This allows for the cells that take up the plasmid to grow and to give the cells time to create the
antibiotic-resistant genes needed.
ii. SOC has no antibiotics but has glucose to support increased growth.
10. While waiting, prepare 50 ml of the following buffer for lysis.
HO lysis buffer: 50mM Tris buffer pH 8, with 150mM NaCl, 5mM EDTA, 2mM DTT, 20% glycerol
Show your calculations to your TA before you weigh the ingredients.

11. Prepare 1L ZY Media by following the recipe (place in plastic bags to store) for the next experiment.
1. 10 g Tryptone
2. 5 g Yeast Extract
12. After the 1 hr. incubation, plate 10 μL, and 50 μL of cells onto pre-warmed plates. Also, run a control of plain
SOC media.
13. As done in the video, use a sterile spreader to spread cells onto the plate.
14. Place plates into 37C incubator O/N
i. Upside down to avoid condensation
15. Come in the next day to check for growth.
16. If growth is present, parafilm and place into 4C fridge for next week.
i. Do not forget to take pictures of plates.